Bronner, M.E. In the early embryo, the neural plate border region contributes to diverse cell fates, ranging from neural crest cells and ectodermal placode cells to neurons of the central nervous system. Despite extensive studies of the neural crest and ectodermal placodes at post-neurula stages, surprisingly little is known about how these populations become distinct from one another within the early neural plate border. Based on our preliminary data, we hypothesize that many neural plate border cells are multipotent as evidenced by their concomitant expression of markers characteristic of several fates. We will test this hypothesis by: 1) conducting a detailed analysis of the emerging neural plate border region by multiplex protein and gene expression profiling coupled with cell lineage analysis and 2) examining how perturbation of transcription factor levels affects expression profiles and lineage allocations of individual neural plate border cells. The significance of this proposal is that it will be the first to test how and when ectodermal placode precursors are segregated from neural crest and neural precursors at the neural plate border. The following aims will be performed: Aim 1: High resolution analysis of protein expression of neural plate, neural crest, placode and other ectodermal markers in the neural plate border as a function of time. We will examine co-expression of transcription factors associated with neural crest, placode, neural plate and ectodermal lineages quantitatively and at single cell resolution in chick gastrula to neurula stages to determine their degree of overlap and if/when a discrete separation occurs between them in the neural plate border. To take this to a multiplex level, we will then perform single molecule fluorescent in situ hybridization analysis (smFISH) at similar stages with 35 or more probes selected from known genes and new candidates from our single cell RNA-seq dataset. Aim 2: Molecular dissection of regulatory interactions that mediate gene expression and cell fate choice at the neural plate border. We will examine the consequence of perturbing individual transcription factors (e.g. Pax7, Sox2, Six1) on expression of others neural plate border genes at the population and single cell level. To examine inputs that regulate neural plate border formation, we will dissect novel enhancers for Pax7, Six1 and other genes to determine direct regulatory inputs. Finally, we will examine how balancing levels of transcription factors may influence other factors in the neural plate border region. Aim 3: Single cell lineage analysis of cells at the neural plate border. To definitively test whether individual cells at the neural plate border have restricted or broad developmental potential, we will carry out single cell lineage analysis by performing iontophoretic injection of lysinated rhodamine dextran into individual neural plate border cells. We also will use enhancers for Pax7, Sox2, or Six1 as well as photoconversion of individual cells to follow the long term fate of neural plate border cells and examine how blocking individual transcription factors affects cell lineage allocation. 1